1. Field
Methods and compositions for modifying myostatin expression. In particular, methods and compositions which inhibit myostatin expression to increase muscle mass, prevent muscle loss and/or ameliorate fibrosis.
2. Background
Myostatin growth differentiation factor-8 (GDF-8 or myostatin) is a member of the transforming growth factor-beta (TGF-beta (β)) superfamily that negatively regulates muscle growth. (Patel K et. al., Neuromuscul Disord. 15:117-126 (2005)). The TGF-beta superfamily includes inhibins, activin, anti-müllerian hormone, and bone morphogenetic protein. TGF-beta acts to control proliferation, differentiation, and other functions in most cell types.
Genetic deletion (i.e. knock-out or KO) or inactivating mutations of myostatin in mice has been shown to result in excessive muscle growth measured by an increase in the size of existing muscle fibers (hypertrophy) and in genetic inactivation of myostatin expression in some mouse models measured by an additional increase in muscle number (hyperplasia). (McPherron et. al., Nature 387: 83-90 (1997)). Moreover, myostatin KO mice have exhibited increased proliferation of myoblasts that ultimately terminally differentiate and donate nuclei to myofibers. (Thomas et al., J Biol Chem 275: 40235-40243 (2000)). In both the myostatin KO mouse model and in naturally occurring bovine mutant cattle breeds (Belgian Blue, Piedmontese, and others), loss of myostatin activity has been shown to increase muscle mass. (Patel et. al., Neuromuscul Disord. 15:117-126 (2005)). A similar ‘muscling’ phenotype has further been documented in a child with inactivating mutations for both myostatin alleles. (Schuelke et al, N Engl J Med 350: 2682-2688 (2004)). Conversely, myostatin protein over-expression has been shown to decrease muscle mass and fiber size in a transgenic mouse model. (Reisz-Porszasz et al, Am J Physiol Endocrinol Metab 285: E876-E888 (2003)).
In addition to its role as a negative regulator of muscle mass, myostatin has recently been implicated in fibrosis. In particular, in vivo long-term administration of myostatin antibodies to an mdx mouse, a model of muscle dystrophy, has been shown to not only enhance myofiber regeneration but also reduce the considerable muscle fibrosis observed in these animals. (Bogdanovich et. al., Nature 420: 418-421 (2002)). Additionally, the myostatin KO mouse shows improved muscle healing and reduced fibrosis in an acute muscle injury model (McCroskery et al., J Cell Sci 118: 3531-3541 (2005) and an mdx-myostatin null double mutant model. (Wagner et. al., Ann Neural 52: 832-836 (2002)).
In view of these findings, attempts have been made to increase muscle mass by controlling the expression of myostatin systemically. In one study, injections of a protease resistant propeptide form of myostatin, which cannot be processed into the putative mature myostatin but can bind the one already produced, were administered weekly over the period of one month. (Wolfman et al, Proc Natl Acad Sci USA 100: 15842-15846 (2003)). As a result of the weekly injections, muscle mass increased by 25 percent (%). (Wolfman et al, Proc Natl Acad Sci USA 100: 15842-15846 (2003)). In another study, daily injections of anti-myostatin antibodies were administered into the peritoneum for one month resulting in a muscle mass increase of 20%. (Whittemore et al, Biochem Biophys Res Commun 300: 965-971 (2003).
Although these studies support the notion that systemically blocking myostatin activity might be therapeutically efficacious in humans, the blockade during embryonic development is associated with substantial side effects. The Belgian Blue cattle and other mutant breeds demonstrate reduced female fertility and an increase in caesarian section birthing due to a decreased ability to carry a fetus to term. (Whittemore et al, Biochem Biophys Res Commun 300: 965-971 (2003)). In addition, these animals abnormally overheat during exercise, a process associated with the excessive musculature. (Chupin D., Analysis of reproduction problems in double muscle females. In: King J W B, Menissier, F. editors. Muscle hypertrophy of genetic origin and its use to improve beef production). Transgenic myostatin KO mice also exhibit some decreased fertility. (Patel K et. al., Neuromuscul Disord. 15:117-126 (2005)).
Still further, there are several problems associated with the general inhibition of myostatin activity by antibodies. In particular, such techniques may be difficult to sustain long term, may lead to undesirable immune responses, and may affect tissues other than the muscle, where an endogenous basal production of myostatin could still be functionally significant.
Delivery of gene constructs into muscle using viral and plasmid based protocols as well as the direct injection of naked DNA has further been documented. Viral based protocols have utilized adeno-associated viruses to express microdystrophin complimentary DNA (cDNA) and helper-dependent adenoviruses to express the full dystrophin gene. Direct injection of naked DNA into muscle without the use of an adjuvant to facilitate uptake of DNA into cells, however, has been found to be relatively poor, possibly due to DNA uptake requiring slow endocytotic mechanisms.